Small animal or laboratory animal research is a cornerstone of modern biomedical advancement. Research using small animals enables researchers to understand complex biological mechanisms, to understand human and animal disease progression, and to develop new drugs to cure or alleviate many human and animal maladies. Small animal research is important in many areas of biomedical research including neurobiology, developmental biology, cardiovascular research and cancer biology. Cardiovascular disease and cancer are currently two of the most common causes of death and morbidity in our society. Therefore, it is extremely important for small animal research to be sufficiently sophisticated and efficient to allow for medical advance in these and other categories of disease.
For small animal research to continue to advance the understanding of diseases, it is of great benefit for researchers to image or visualize structures within a small animal. Structures within the small animal that could benefit from imaging include, but are not limited to, tissues, organs, and cavities. Moreover, it is valuable for these structures to be imaged longitudinally over an animal's lifetime. One method for visualizing structures within a small animal is invasive surgery. Invasive surgery involves surgically invading an animal to visualize its internal structures. Once the animal is incised and the desired structure visualized, the animal's incision can be closed and the animal can be allowed to recover, or the animal can be sacrificed. If a researcher wants to visualize the same structure at a subsequent time in the animal's lifetime, surgery can be repeated on the same animal, or, if the small animal was sacrificed, a different animal can be visualized at the desired period in its life. Invasive surgery, however, has many drawbacks.
The drawbacks of invasive surgery include, but are not limited to, poor results, potential surgical complications and high costs. Results obtained using surgery are often poor because surgery can stress the animal or cause post surgical complications, including infection. An animal's stress response to surgery may prevent a researcher from drawing accurate conclusions regarding that animal's response to a given disease, drug, or medical procedure. Post surgical infection can also negatively affect results. Moreover, an infection can kill the animal, or require the animal to be medically treated or sacrificed. If an animal dies from infection, another post surgical complication, or is sacrificed, another animal must be studied. When a different animal must be used, inaccuracies are inherently introduced into a researcher's findings. These inaccuracies may be due to individual differences between study animals, differing husbandry conditions, or any other number of potential differences. All of these drawbacks increase the cost of research by increasing the number of animals needed and by making poor results more likely.
Non-invasive ultrasound has long been used as a diagnostic tool to aid in therapeutic procedures. It is based on the principle that waves of sound energy can be focused upon an area of interest, reflected and processed to produce an image. To improve the images obtained using conventional, or low frequency ultrasound, echogenic contrast agents are sometimes used to create a reflector of ultrasonic energy in an area of interest. In conventional frequency ultrasound, a rapid development of microbubble contrast imaging techniques for medical ultrasound has occured. Non-linear scattering from resonant bubble population has been exploited to implement a variety of detection methods, which are used to suppress tissue signals and enhance the detection of blood. Non-linear microbubble imaging for commercial ultrasound operating at conventional frequencies has demonstrated important clinical utility in improving structure visualization including improving small vessel detection and in cardiac chamber imaging. Because conventional frequency ultrasound operates in the 1-8 MHz range, microbubble contrast agents have been designed to work well within this frequency range.
Ultrasound has recently been adapted for use in small animal research. In particular, high frequency ultrasound has been used to visualize anatomical structures and hemodynamic function in longitudinal studies of small animals. High frequency ultrasound imaging of small animals is non-invasive and allows longitudinal studies of individual animals. These studies reduce the number of animals required for analysis and alleviate many problems associated with invasive surgery. Potential areas of small animal research where high frequency ultrasound imaging is beneficial include, but are not limited to, cancer and angiogenesis studies, developmental biology, cardiovascular research and neurological research. In each of these areas, ultrasound imaging offers the distinct advantage of non-invasive longitudinal studies that were previously unavailable using invasive surgery or conventional frequency ultrasound.
To improve on these advantages it would be desirable to take advantage of non-linear scattering by microbubble contrast agents of high frequency ultrasound in small animals. Improved high frequency ultrasound imaging in small animals using microbubble contrast agents could improve the sophistication and efficiency of biomedical research and drug development in small animals. The need exists in the art for a method of producing an ultrasound image of a small animal and its internal structures that is enhanced by non-linear scattering of ultrasound by microbubble contrast agents. A need also exists for a composition of microbubbles designed to enhance imaging when high-frequency ultrasound is utilized.